Modified nucleosides and nucleotides and uses thereof

ABSTRACT

The invention is directed to modified guanine-containing nucleosides and nucleotides and uses thereof. More specifically, the invention relates to modified fluorescently labelled guanine-containing nucleosides and nucleotides which exhibit enhanced fluorophore intensity by virtue of reduced quenching effects.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority from Great Britain ApplicationSerial No. 0517097.2, filed on Aug. 19, 2005. Applicants claim priorityunder 35 U.S.C. § 119 as to the said Great Britain application, and theentire disclosure of said application is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The invention relates to modified guanine-containing nucleosides andnucleotides and more specifically to modified fluorescently labelledguanine-containing nucleosides and nucleotides which exhibit reducedquenching effects, and hence enhanced brightness of the fluorophore.

BACKGROUND TO THE INVENTION

Advances in the study of biological molecules have been led, in part, byimprovement in technologies used to characterise the molecules or theirbiological reactions. In particular, the study of the nucleic acids DNAand RNA has benefited from developing technologies used for sequenceanalysis.

Nucleic acid sequencing methods have been known in the art for manyyears. One of the best-known methods is the Sanger “dideoxy” methodwhich relies upon the use of dideoxyribonucleoside triphosphates aschain terminators. The Sanger method has been adapted for use inautomated sequencing with the use of chain terminators incorporatingfluorescent labels.

There are also known in the art methods of nucleic acid sequencing basedon successive cycles of incorporation of fluorescently labelled nucleicacid analogues. In such “sequencing by synthesis” or “cycle sequencing”methods the identity of the added base is determined after eachnucleotide addition by detecting the fluorescent label.

In particular, U.S. Pat. No. 5,302,509 describes a method for sequencinga polynucleotide template which involves performing multiple extensionreactions using a DNA polymerase to successively incorporate labelledpolynucleotides complementary to a template strand. In such a“sequencing by synthesis” reaction a new polynucleotide strandbased-paired to the template strand is built up in the 5′ to 3′direction by successive incorporation of individual nucleotidescomplementary to the template strand. The substrate nucleosidetriphosphates used in the sequencing reaction are labelled at the 3′position with different 3′ labels, permitting determination of theidentity of the incorporated nucleotide as successive nucleotides areadded.

The guanine base of DNA is known to act as a quencher of somefluorophores, meaning that a fluorophore attached to G is harder todetect than the equivalent fluorophore attached to C, A or T (Torimuraet al., Analytical Sciences, 17: 155-160 (2001); Kutata et al., NucleicAcids Res., 29(6) e34 (2001)). In the context of a sequencing reactionbased on detection of fluorescent labelled nucleotides, this in turnmeans that the fluorescent signal detected from labelled guaninenucleotides incorporated during the sequencing reaction will be of lowerintensity than that detected from labelled nucleotides bearing the samefluorophore attached to adenine, thymine or cytosine containingnucleotides. Thus, in certain circumstances the presence of a “G”nucleotide may be harder to call with certainty than the presence of A,T or C under the same reaction and detection conditions.

Accordingly, in the context of nucleic acid sequencing reactions itwould be desirable to be able to increase the intensity of thefluorescent signal from fluorescently labelled G nucleotides so that theintensity of the signal compares more favourably with that which can beobtained from fluorescently labelled A, T or C nucleotides under thesame reaction and detection conditions.

SUMMARY OF THE INVENTION

The inventors have now determined that by altering, and in particularincreasing, the length of the linker between the fluorophore and theguanine base, so as to introduce a polyethylene glycol spacer group, itis possible to increase the fluorescence intensity compared to the samefluorophore attached to the guanine base through prior art linkages. Thedesign of the linkers, and especially their increased length, alsoallows improvements in the brightness of fluorophores attached to theguanine bases of guanosine nucleotides when incorporated intopolynucleotides such as DNA. The nucleotides of the invention are thusof use in any method of analysis which requires detection of afluorescent label attached to a guanine-containing nucleotide, includingbut not limited to nucleic acid sequencing and nucleic acid labelling.

Therefore, in a first aspect the invention provides a modifiednucleotide or nucleoside comprising a guanine base or a derivativethereof attached to a fluorophore through a linking group, characterisedin that said linking group comprises a spacer group of formula—((CH₂)₂O)_(n)— wherein n is an integer between 2 and 50.

In a second aspect the invention provides a polynucleotide comprising atleast one modified nucleotide according to the first aspect of theinvention.

In a third aspect the invention provides use of a modified nucleotide ornucleoside according to the first aspect of the invention or apolynucleotide according to the second aspect of the invention in anymethod of analysis which requires detection of a fluorescent signal fromthe modified nucleotide or nucleoside.

In particular embodiments the invention provides use of a modifiednucleotide or nucleoside according to the first aspect of the inventionor a polynucleotide according to the second aspect of the invention in amethod of nucleic acid sequencing, re-sequencing, whole genomesequencing, single nucleotide polymorphism scoring, or any otherapplication involving the detection of the modified nucleotide ornucleoside when incorporated into a polynucleotide.

In a further aspect the invention provides a method of detecting amodified guanosine nucleotide incorporated into a polynucleotide whichcomprises:

(a) incorporating at least one modified nucleotide according to thefirst aspect of the invention into a polynucleotide and

(b) detecting the modified nucleotide(s) incorporated into thepolynucleotide by detecting the fluorescent signal from said modifiednucleotide(s).

In a preferred embodiment the at least one modified nucleotide isincorporated into a polynucleotide by the action of a polymerase enzyme.

In a particular embodiment step (a) may comprise incubating a templatepolynucleotide strand with a reaction mixture comprising fluorescentlylabelled modified nucleotides according to the first aspect of theinvention and a polymerase under conditions which permit formation of aphosphodiester linkage between a free 3′ hydroxyl group on apolynucleotide strand annealed to said template polynucleotide strandand a 5′ phosphate group on said modified nucleotide.

Specific but non-limiting embodiments of this method compriseincorporation of modified nucleotides according to the invention byinter alia polymerase chain reaction (PCR), primer extension, nicktranslation or strand displacement polymerisation.

In a still further aspect, the invention provides a method of sequencinga template nucleic acid molecule comprising:

incorporating one or more nucleotides into a strand of nucleic acidcomplementary to the template nucleic acid and determining the identityof the base present in one or more incorporated nucleotide(s) in orderto determine the sequence of the template nucleic acid molecule;

wherein the identity of the base present in said nucleotide(s) isdetermined by detecting a fluorescent signal produced by saidnucleotide(s);

characterised in that at least one incorporated nucleotide is a modifiednucleotide according to the first aspect of the invention.

In a still further aspect, the invention provides a kit comprising aplurality of different nucleotides including a modified nucleotideaccording to the first aspect of this invention.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph evidencing the improved brightness of the Alexa 488fluorophore in modified nucleotides of the invention having—((CH₂)₂O)₁₁— (denoted G-PEG12-A488) and —((CH₂) 2O)₂₃— (denotedG-PEG24-A488) spacing groups over a modified nucleotide not of thisinvention with no such spacer (denoted G-N₃-A488, and improvedbrightness of the fluorophore having the —((CH₂)₂O)₂₃—, as opposed tothe —((CH₂)₂O)₁₁—, spacing group. Fluorescence intensity was measuredfor each labelled nucleotide in 100 mM Tris, 30 mM NaCl pH7 whenincorporated into polynucleotide both before and after treatment withTCEP to cleave the linking group. Cleavage of the linkers with TCEPshows that the free fluorophore is not quenched in solution, thus theenhanced signal is not simply caused by the PEG moiety attached to thefluorophore.

DETAILED DESCRIPTION

The present invention will now be further described. In the followingpassages, different aspects of the invention are defined in more detail.Each aspect so defined may be combined with any other aspect or aspectsunless clearly indicated to the contrary. In particular, any featureindicated as being preferred or advantageous may be combined with anyother feature or features indicated as being preferred or advantageous.

When describing the invention, certain terms used have particularmeanings to those skilled in the art some of which are as set forthbelow. These definitions are to be used in construing the terms unlessthe context dictates otherwise.

The invention, as described and claimed herein, provides improvedmodified guanosine nucleosides and nucleotides, methods of using these,particularly methods of using guanosine nucleotides in molecularbiological applications where it is desired to monitor incorporation ofthe modified nucleotides into polynucleotides, including but not limitedto sequencing by synthesis applications and other applications involvinglabelling of nucleic acids, and kits comprising such nucleosides ornucleotides.

As is known in the art, a “nucleotide” consists of a nitrogenous base, asugar, and one or more phosphate groups. “Nucleosides” consist of thenitrogenous base and sugar only. In naturally occurring or nativenucleotides the sugar component is usually either ribose, as inribonucleotides and the corresponding polynucleotide RNA, ordeoxyribose, i.e., a sugar lacking the 2′ hydroxyl group that is presentin ribose, as in deoxyribonucleotides and the correspondingpolynucleotide DNA. The naturally occurring sugars may be modified, forexample by removal or substitution of the 3′ hydroxyl group. Thenitrogenous base is a derivative of purine or pyrimidine. The purinesare adenine (A) and guanine (G), and the pyrimidines are cytosine (C)and thymine (T) (or in the context of RNA, uracil (U)). The equivalentnucleosides incorporating these bases are respectively denotedadenosine, guanosine, cytidine and thymidine. The C-1 atom ofdeoxyribose is bonded to N-1 of a pyrimidine or N-9 of a purine. Anucleotide is also a phosphate ester of a nucleoside, withesterification occurring on the hydroxyl group attached to C-5 of thesugar. Nucleotides may be mono, di, tri or cyclic phosphates.

The modified nucleotides or nucleosides of the invention comprise theguanine base, sugar (and one or more phosphate groups, if appropriate)and a detectable label comprising a fluorophore. The detectable label isattached to the guanine base through a linking group.

In the modified nucleosides and nucleotides of the invention the linkinggroup comprises a polyethylene glycol spacer, although other suitablyhydrophilic groups of similar length to the polyethylene glycol spacersof the invention (approximately 5 to 150 atoms) may be used as analternative to polyethylene glycol spacers. Preferably the spacercomprises between 5 and 30 ethylene oxide groups —((CH₂)₂O)—, still morepreferably between 10 and 25 ethylene oxide groups. Exemplified herein,and thus particularly preferred ethylene oxide spacer groups are—((CH₂)₂O)₁₁— and —((CH₂)₂O)₂₃—, preferably —((CH₂)₂O)₂₃—.

The linking groups used in the present invention serve to space thefluorescent label away from the guanine base such that the amount ofquenching of the fluorescent signal from the fluorophore by the guaninebase is reduced or substantially eliminated, as compared to a nucleotideor nucleoside of analogous structure but lacking the linking group. Atthe same time, the fluorophore is maintained in indirect covalentattachment with the remainder of the nucleotide/nucleoside.

The nature of the fluorophore present in the fluorescent label isgenerally not limited. It may be any fluorophore compatible withlabelling of nucleosides/nucleotides and, depending on the intended useof the modified nucleotides, also with subsequent incorporation of themodified nucleotides into a polynucleotide. The invention isparticularly applicable to modified nucleotides labelled with anyfluorophore that shows a decrease in the fluorescence emission intensitywhen covalently attached to a guanosine nucleotide. Appropriatefluorophores are well known to those skilled in the art and may beobtained from a number of commercial manufacturers, such as MolecularProbes Inc.

For example, Welch et al. (Chem. Eur. J. 5(3):951-960, 1999) disclosesdansyl-functionalised fluorescent moieties that can be used in thepresent invention. Zhu et al. (Cytometry 28:206-211, 1997) describes theuse of the fluorescent labels Cy3 and Cy5, which can also be used in thepresent invention. Labels suitable for use are also disclosed in Proberet al. (Science 238:336-341, 1987); Connell et al. (BioTechniques5(4):342-384, 1987), Ansorge et al. (Nucl. Acids Res. 15(11):4593-4602,1987) and Smith et al. (Nature 321:674, 1986). Other commerciallyavailable fluorescent labels include, but are not limited to,fluorescein, rhodamine (including TMR, texas red and Rox), alexa,bodipy, acridine, coumarin, pyrene, benzanthracene and the cyanins.

For example, two classes of particularly preferred fluorophores whichmay be used according to this invention are the Alexa series availablefrom Molecular Probes, (sometimes referred to as Alexa Fluor dyes) andfluorescent labels in the Atto series available from Atto-tec (sometimesreferred to as Atto fluorescent labels) of Atto-tec. An example of apreferred Alexa dye is Alexa 488, and an example of a particular Attodye is Atto 532.

Other than the ethylene oxide spacer moiety, the linkage between thebase and detectable label may comprise other chemical functionality.This may serve to afford cleavable or non-cleavable linkers. Examples ofcleavable linkers are known to those skilled in the art (see for exampleApplicant's published International patent applications WO03/048387 andWO2004/018493).

As aforesaid, the sugar components of the nucleosides and nucleotidesaccording to the invention may be further modified (from the nativeribose or deoxyribose) in order to confer some useful property withoutaffecting the function of the fluorescent label component. Aparticularly preferred embodiment of the invention is the provision ofmodified guanosine nucleosides and nucleotides having a cleavable 3′blocking group, and most preferably deoxyribonucleosides anddeoxyribonucleotides including such a 3′ blocking group. Exemplary, andpreferred, blocking groups are described in our co-pending applicationWO 2004/018497. Preferably the nucleoside and nucleotides of theinvention contain a 3′ blocking group and a cleavable linker to thedetectable label, more preferably still wherein the block and linker mayboth be cleaved under the same conditions so as to reveal the 3′-OHgroup in the resultant product upon a single chemical reaction. Examplesof such functionalities are described fully in WO2004/018497.

Linkage of the fluorescent label to the guanine base via the linkinggroup may be to any suitable position of the base, provided that it doesnot interfere with the intended function/use of the modified nucleotideor nucleoside. For example, if a modified nucleotide according to theinvention is to be enzymatically incorporated into a polynucleotide bythe action of a polymerase then the position of linkage of thefluorescent label via the linking group should not prevent suchenzymatic incorporation. Typically linkage will be via the 7 position ofthe “guanine” base. It will be appreciated that in order to provide thenecessary valency for covalent linkage at the 7 position a 7-deazaguanine derivative may be used in preference to the native guanine base.Accordingly, references herein to modified “guanine-containing”nucleosides or nucleotides or to modified “guanosine” nucleosides ornucleotides should be interpreted as encompassing analogous structureswhich contain a guanine derivative, and in particular 7-deaza guanine,rather than the native guanine base, unless the context impliesotherwise. In other embodiments the linking group may be attached to the8 position of the guanine ring system. Further modifications orsubstitutions may be included elsewhere in the guanine ring system, inaddition to the position at which the linking group is attached, as infor example 7-deaza-8-aza guanine. Again references herein to modified“guanine-containing” nucleosides or nucleotides or to modified“guanosine” nucleosides or nucleotides should be interpreted asencompassing such further modified forms of the guanine base unless thecontext implies otherwise.

In specific, but non-limiting, embodiments described herein withreference to the accompanying examples the invention provides:

-   7-[3-(-Alexa488-PEG₁₂-LN₃-linker    acetylamino)-prop-1-ynyl]-3′-azidomethyl-dGTP, and-   7-[3-(-Alexa488-PEG₂₄-LN₃-linkeracetylamino)-prop-1-ynyl]-3′-azidomethyl-dGTP

The invention also encompasses polynucleotides incorporating one or moremodified guanosine nucleotides according to the invention. Preferablysuch polynucleotides will be DNA or RNA, comprised respectively ofdeoxyribonucleotides or ribonucleotides joined in phosphodiesterlinkage. Polynucleotides according to the invention may comprisenaturally occurring nucleotides, non-natural (or modified) nucleotidesother than the modified nucleotides of the invention or any combinationthereof, provided that at least one modified nucleotide according to theinvention is present. Polynucleotides according to the invention mayalso include non-natural backbone linkages and/or non-nucleotidechemical modifications. Chimeric structures comprised of mixtures ofribonucleotides and deoxyribonucleotides are also contemplated.

Preferred Uses of the Nucleotides of the Invention

The modified nucleotides (or nucleosides) of the invention may be usedin any method of analysis which requires detection of a fluorescentlabel attached to a guanine-containing nucleotide or nucleoside, whetheron its own or incorporated into or associated with a larger molecularstructure or conjugate. In all such methods of analysis the use of themodified guanosine nucleotides or nucleosides of the invention providesan advantage in that the brightness of the fluorescent signal isincreased compared to that which would be obtained using guanosinenucleotides or nucleosides of analogous structure but lacking the longerlinking group present in the modified nucleotides or nucleosides of theinvention.

In particular embodiments of the invention, modified nucleotides of theinvention may be used in any method of analysis which requires detectionof a fluorescent label attached to a modified guanine nucleotideincorporated into a polynucleotide. In this context the term“incorporated into a polynucleotide” requires that the 5′ phosphate isjoined in phosphodiester linkage to the 3′ hydroxyl group of a second(modified or unmodified) nucleotide, which may itself form part of alonger polynucleotide chain. The 3′ end of the modified nucleotide ofthe invention may or may not be joined in phosphodiester linkage to the5′ phosphate of a further (modified or unmodified) nucleotide.

Thus, in one non-limiting embodiment the invention provides a method ofdetecting a modified guanosine nucleotide incorporated into apolynucleotide which comprises:

(a) incorporating at least one modified nucleotide according to thefirst aspect of the invention into a polynucleotide and

(b) detecting the modified nucleotide(s) incorporated into thepolynucleotide by detecting the fluorescent signal from said modifiednucleotide(s).

This method requires two essential steps: a synthetic step (a) in whichone or more modified nucleotides according to the invention areincorporated into a polynucleotide and a detection step (b) in which oneor more modified nucleotide(s) incorporated into the polynucleotide aredetected by detecting or quantitatively measuring their fluorescence.

In a preferred embodiment the at least one modified nucleotide isincorporated into a polynucleotide in the synthetic step by the actionof a polymerase enzyme. However, other methods of joining modifiednucleotides to polynucleotides, such as for example chemicaloligonucleotide synthesis, are not excluded. Therefore, in the specificcontext of this method of the invention, the term “incorporating” anucleotide into a polynucleotide encompasses polynucleotide synthesis bychemical methods as well as enzymatic methods.

In a specific embodiment the synthetic step may comprise incubating atemplate polynucleotide strand with a reaction mixture comprisingfluorescently labelled modified guanosine nucleotides of the inventionand a polymerase under conditions which permit formation of aphosphodiester linkage between a free 3′ hydroxyl group on apolynucleotide strand annealed to said template polynucleotide strandand a 5′ phosphate group on said modified nucleotide.

This embodiment comprises a synthetic step in which formation of apolynucleotide strand is directed by complementary base-pairing ofnucleotides to a template strand.

In all embodiments of the method, the detection step may be carried outwhilst the polynucleotide strand into which the modified guanosinenucleotides are incorporated is annealed to a template strand, or aftera denaturation step in which the two strands are separated. Furthersteps, for example chemical or enzymatic reaction steps or purificationsteps, may be included between the synthetic step and the detectionstep. In particular, the target strand incorporating the modifiednucleotide(s) may be isolated or purified and then processed further orused in a subsequent analysis. By way of example, target polynucleotideslabelled with modified nucleotide(s) according to the invention in asynthetic step may be subsequently used as labelled probes or primers.In other embodiments the product of the synthetic step (a) may besubject to further reaction steps and, if desired, the product of thesesubsequent steps purified or isolated.

Suitable conditions for the synthetic step will be well known to thosefamiliar with standard molecular biology techniques. In one embodimentthe synthetic step may be analogous to a standard primer extensionreaction using nucleotide precursors, including modified guanosinenucleotides according to the invention, to form an extended targetstrand complementary to the template strand in the presence of asuitable polymerase enzyme. In other embodiments the synthetic step mayitself form part of a polymerase chain reaction producing a labelleddouble-stranded PCR product comprised of annealed complementary strandsderived from copying of the target and template polynucleotide strands.Other exemplary “synthetic” steps include nick translation, stranddisplacement polymerisation, random primed DNA labelling etc.

The polymerase enzyme used in the synthetic step must be capable ofcatalysing the incorporation of modified guanosine nucleotides accordingto the invention. Otherwise, the precise nature of the polymerase is notparticularly limited but may depend upon the conditions of the syntheticreaction. For example, if the synthetic reaction is a PCR reaction thena thermostable polymerase is required, whereas this is not essential forstandard primer extension. Suitable thermostable polymerases which arecapable of incorporating the modified nucleotides according to theinvention include those described in WO 2005/024010.

In specific non-limiting embodiments the invention encompasses use ofthe modified nucleotides or nucleosides according to the invention in amethod of nucleic acid sequencing, re-sequencing, whole genomesequencing, single nucleotide polymorphism scoring, any otherapplication involving the detection of the modified nucleotide ornucleoside when incorporated into a polynucleotide, or any otherapplication requiring the use of polynucleotides labelled with thefluorescent modified nucleotides according to the invention.

In a particularly preferred embodiment the invention provides use ofmodified nucleotides according to the invention in a polynucleotide“sequencing-by-synthesis” reaction. Sequencing-by-synthesis generallyinvolves sequential addition of one or more nucleotides to a growingpolynucleotide chain in the 5′ to 3′ direction using a polymerase inorder to form an extended polynucleotide chain complementary to thetemplate nucleic acid to be sequenced. The identity of the base presentin one or more of the added nucleotide(s) is determined in a detectionor “imaging” step. The identity of the added base is preferablydetermined after each nucleotide incorporation step. The sequence of thetemplate may then be inferred using conventional Watson-Crickbase-pairing rules. For the avoidance of doubt “sequencing” can alsoencompass incorporation and identification of a single nucleotide.Determination of the identity of a single base may be useful, forexample, in the scoring of single nucleotide polymorphisms.

In nucleic acid sequencing protocols, because the brightness of thefluorescent signal obtained from the modified nucleotides of theinvention is increased compared to that which would be obtained usingguanosine nucleotides of analogous structure but lacking the longerlinking group present in the modified nucleotides or nucleosides of theinvention, it is possible to “call” the presence of G nucleotidesaccurately at much lower template concentrations. With prior artguanosine nucleotides the brightness of the fluorescence from G may be alimiting factor on the performance of any given sequencing reaction,particularly affecting the lower limit on the amount of template whichmust be added to the reaction. With the use of the modified nucleotidesof the invention the amount of fluorescence from each individualincorporated guanosine nucleotide is increased, hence it may be possibleto accurately sequence reduced amounts of template.

In an embodiment of the invention, the sequence of a templatepolynucleotide is determined in a similar manner to that described inU.S. Pat. No. 5,654,413, by detecting the incorporation of one or morenucleotides into a nascent strand complementary to the templatepolynucleotide to be sequenced through the detection of fluorescentlabel(s) attached to the incorporated nucleotide(s). Sequencing of thetemplate polynucleotide is primed with a suitable primer (or prepared asa hairpin construct which will contain the primer as part of thehairpin), and the nascent chain is extended in a stepwise manner byaddition of nucleotides to the 3′ end of the primer in apolymerase-catalysed reaction.

In preferred embodiments each of the different nucleotides (A, T, G andC) is labelled with a unique fluorophore which acts as a blocking groupat the 3′ position to prevent uncontrolled polymerisation. Thepolymerase enzyme incorporates a nucleotide into the nascent chaincomplementary to the template polynucleotide, and the blocking groupprevents further incorporation of nucleotides. Any unincorporatednucleotides are removed and each incorporated nucleotide is “read”optically by suitable means, such as a charge-coupled device using laserexcitation and filters. The 3′-blocking group is then removed(deprotected), to expose the nascent chain for further nucleotideincorporation. Typically the identity of the incorporated nucleotidewill be determined after each incorporation step but this is notstrictly essential.

Similarly, U.S. Pat. No. 5,302,509 discloses a method to sequencepolynucleotides immobilised on a solid support. The method relies on theincorporation of fluorescently-labelled, 3′-blocked nucleotides A, G, Cand T into a growing strand complementary to the immobilisedpolynucleotide, in the presence of DNA polymerase. The polymeraseincorporates a base complementary to the target polynucleotide, but isprevented from further addition by the 3′-blocking group. The label ofthe incorporated base can then be determined and the blocking groupremoved by chemical cleavage to allow further polymerisation to occur.

The nucleic acid template to be sequenced in a sequencing-by-synthesisreaction may be any polynucleotide that it is desired to sequence. Thenucleic acid template for a sequencing reaction will typically comprisea double-stranded region having a free 3′ hydroxyl group which serves asa primer or initiation point for the addition of further nucleotides inthe sequencing reaction. The region of the template to be sequenced willoverhang this free 3′ hydroxyl group on the complementary strand. Theoverhanging region of the template to be sequenced may be singlestranded but can be double-stranded, provided that a “nick is present”on the strand complementary to the template strand to be sequenced toprovide a free 3′ OH group for initiation of the sequencing reaction. Insuch embodiments sequencing may proceed by strand displacement. Incertain embodiments a primer bearing the free 3′ hydroxyl group may beadded as a separate component (e.g. a short oligonucleotide) whichhybridises to a single-stranded region of the template to be sequenced.Alternatively, the primer and the template strand to be sequenced mayeach form part of a partially self-complementary nucleic acid strandcapable of forming an intramolecular duplex, such as for example ahairpin loop structure. Preferred hairpin polynucleotides and methods bywhich they may be attached to solid supports are disclosed in ourco-pending International application publication no. WO 2005/047301.

Nucleotides are added successively to the free 3′ hydroxyl group,resulting in synthesis of a polynucleotide chain in the 5′ to 3′direction. The nature of the base which has been added may bedetermined, preferably but not necessarily after each nucleotideaddition, thus providing sequence information for the nucleic acidtemplate.

The term “incorporation” of a nucleotide into a nucleic acid strand (orpolynucleotide) refers to joining of the nucleotide to the free 3′hydroxyl group of the nucleic acid strand via formation of aphosphodiester linkage with the 5′ phosphate group of the nucleotide.

The nucleic acid template to be sequenced may be DNA or RNA, or even ahybrid molecule comprised of deoxynucleotides and ribonucleotides. Thenucleic acid template may comprise naturally occurring and/ornon-naturally occurring nucleotides and natural or non-natural backbonelinkages, provided that these do not prevent copying of the template inthe sequencing reaction.

In certain embodiments the nucleic acid template to be sequenced may beattached to a solid support via any suitable linkage method known in theart. Preferably linkage will be via covalent attachment.

In certain embodiments template polynucleotides may be attached directlyto a solid support (e.g. a silica-based support). However, in otherembodiments of the invention the surface of the solid support may bemodified in some way so as to allow either direct covalent attachment oftemplate polynucleotides, or to immobilise the template polynucleotidesthrough a hydrogel or polyelectrolyte multilayer, which may itself benon-covalently attached to the solid support.

Arrays in which polynucleotides have been directly attached tosilica-based supports are those for example disclosed in WO 97/04131,wherein polynucleotides are immobilised on a glass support by reactionbetween a pendant epoxide group on the glass with an internal aminogroup on the polynucleotide. In addition, we disclose in our co-pendingInternational patent application publication number WO2005/047301 arraysof polynucleotides attached to a solid support, e.g. for use in thepreparation of SMAs, or clustered microarrays, by reaction of asulfur-based nucleophile with the solid support.

A still further example of solid-supported template polynucleotides iswhere the template polynucleotides are attached a to hydrogel supportedupon silica-based or other solid supports. Silica-based supports aretypically used to support hydrogels and hydrogel arrays as described inWO00/31148, WO01/01143, WO02/12566, WO03/014392, U.S. Pat. No. 6,465,178and WO00/53812.

A particularly preferred surface to which template polynucleotides maybe immobilised is a polyacrylamide hydrogel. Polyacrylamide hydrogelsare described in the prior art, some of which is discussed above.However, a particularly preferred hydrogel is described in WO2005/065814.

Preferably, where the template polynucleotide is immobilized on or to asolid support, this comprises a planar wave guide as is described in ourco-pending British patent application no. 0507835.7.

The use of a planar wave guide serves to enhance the sensitivity ofdetection of a nucleotide incorporated into a polynucleotide molecule,wherein the incorporated nucleotide is detected by detecting a signalproduced by said nucleotide when exposed to an evanescent fieldgenerated by coupling of light into said planar waveguide.

The template(s) to be sequenced may form part of an “array” on a solidsupport, in which case the array may take any convenient form. Thus, themethod of the invention is applicable to all types of “high density”arrays, including single-molecule arrays and clustered arrays.

The method of the invention may be used for sequencing templates onessentially any type of array formed by immobilisation of nucleic acidmolecules on a solid support, and more particularly any type ofhigh-density array. However, the method of the invention is particularlyadvantageous in the context of sequencing of clustered arrays.

In multi-polynucleotide or clustered arrays distinct regions on thearray comprise multiple polynucleotide template molecules. The term“clustered array” refers to an array wherein distinct regions or siteson the array comprise multiple polynucleotide molecules that are notindividually resolvable by optical means. Depending on how the array isformed each site on the array may comprise multiple copies of oneindividual polynucleotide molecule or even multiple copies of a smallnumber of different polynucleotide molecules (e.g. multiple copies oftwo complementary nucleic acid strands).

Multi-polynucleotide or clustered arrays of nucleic acid molecules maybe produced using techniques generally known in the art. By way ofexample, WO 98/44151 and WO 00/18957 both describe methods of nucleicacid amplification which allow amplification products to be immobilisedon a solid support in order to form arrays comprised of clusters or“colonies” of immobilised nucleic acid molecules. The nucleic acidmolecules present on the clustered arrays prepared according to thesemethods are suitable templates for sequencing using the method of theinvention.

The sequencing method of the invention is also applicable to sequencingof templates on single molecule arrays. The term “single molecule array”or “SMA” as used herein refers to a population of polynucleotidemolecules, distributed (or arrayed) over a solid support, wherein thespacing of any individual polynucleotide from all others of thepopulation is such that it is possible to effect individual resolutionof the polynucleotides. The target nucleic acid molecules immobilisedonto the surface of the solid support should thus be capable of beingresolved by optical means. This means that, within the resolvable areaof the particular imaging device used, there must be one or moredistinct signals, each representing one polynucleotide. This may beachieved, preferably wherein the spacing between adjacent polynucleotidemolecules on the array is at least 100 nm, more preferably at least 250nm, still more preferably at least 300 nm, even more preferably at least350 nm. Thus, each molecule is individually resolvable and detectable asa single molecule fluorescent point, and fluorescence from said singlemolecule fluorescent point also exhibits single step photobleaching. Theterms “individually resolved” and “individual resolution” are usedherein to specify that, when visualised, it is possible to distinguishone molecule on the array from its neighbouring molecules. Separationbetween individual molecules on the array will be determined, in part,by the particular technique used to resolve the individual molecules.The general features of single molecule arrays will be understood byreference to published applications WO 00/60770 and WO 01/57248.

Although a preferred use of the modified nucleotides of the invention isin sequencing-by-synthesis reactions the utility of the modifiednucleotides is not limited to such methods. In fact, the nucleotides maybe used advantageously in any sequencing methodology which requiresdetection of fluorescent labels attached to guanosine nucleotidesincorporated into a polynucleotide.

In particular, the modified nucleotides of the invention may be used anautomated fluorescent sequencing protocols, particularly fluorescentdye-terminator cycle sequencing based on the chain terminationsequencing method of Sanger and co-workers. Such methods generally usePCR to incorporate fluorescently labelled dideoxynucleotides in a primerextension sequencing reaction.

So-called Sanger sequencing methods, and related protocols(Sanger-type), rely upon randomised chain-termination at labeleddideoxynucleotides including a known base. An example of a Sanger-typesequencing protocol is the BASS method described by Metzker (NucleicAcids Research, 22(2)):4259-4267, 1994). Other Sanger-type sequencingmethods will be known to those skilled in the art.

Thus, the invention also encompasses modified guanosine nucleotidesaccording to the invention which are dideoxynucleotides lacking hydroxylgroups at both the 3′ and 2′ positions, such modified dideoxynucleotidesbeing suitable for use in Sanger type sequencing methods. Modifiedguanosine nucleotides of the present invention incorporating 3′ blockinggroups, it will be recognized, may also be of utility in Sanger methodsand related protocols since the same effect achieved by using modifieddideoxyguanosine nucleotides may be achieved by using guanosinenucleotides having 3′-OH blocking groups: both prevent incorporation ofsubsequent nucleotides.

Where nucleotides according to the present invention, and having a 3′blocking group are to be used in Sanger or a Sanger-type sequencingmethods it will be appreciated that the detectable labels attached tothe nucleotides need not be connected via cleavable linkers, since ineach instance where a labeled nucleotide of the invention isincorporated, no nucleotides need to be subsequently incorporated andthus the label need not be removed from the nucleotide.

The invention also provides kits including modified guanosinenucleosides and/or nucleotides according to the invention. Such kitswill generally include a supply of at least one modified nucleotide ornucleoside according to the invention together with at least one furthercomponent. The further component(s) may be further modified orunmodified nucleotides or nucleosides. For example, modified guanosinenucleotides according to the invention may be supplied in combinationwith unlabelled or native guanosine nucleotides, and/or with unlabelledor native adenosine, cytidine or thymidine nucleotides and/or withfluorescently labeled adenosine, cytidine or thymidine nucleotides orany combination thereof. Combinations of nucleotides may be provided asseparate individual components or as nucleotide mixtures In otherembodiments the kits may include a supply of a polymerase enzyme capableof catalyzing incorporation of the modified guanosine nucleotides into apolynucleotide. The polymerase component may be included in addition toor instead of further nucleotide components. Other components to beincluded in such kits may include buffers etc.

The modified nucleotides according to the invention, and other anynucleotide components including mixtures of different nucleotides, maybe provided in the kit in a concentrated form to be diluted prior touse. In such embodiments a supply of a suitable dilution buffer may beincluded.

The invention will be further understood with reference to the followingexperimental examples.

EXAMPLES Example 1 Synthesis of 7-[3-(-Alexa488-PEG₁₂-LN₃-linkeracetylamino)-prop-1-ynyl]-3′-azidomethyl-dGTP (3)

Alexa488-PEG₁₂ carboxylic acid (1)

Alexa fluor 488 carboxylic acid succinimidyl ester (mixed isomers, 20mg, 31 μmoles) was dissolved in dry DMF (1 ml). A solution of aminodPEG₁₂ acid (55.5 mg, 90 μmoles) and DIPEA (31.3 μl, 180 μmoles) in 0.1M triethyl ammonium bicarbonate buffer (TEAB, 0.5 ml, pH 7.5) was added.The reaction was then stirred at RT for 3 hrs. All the reaction mixturewas diluted with 0.1 M TEAB (5 ml) and loaded onto a column of DEAE-A25Sephadex (1×12 cm). The column was eluted with 0.1 M (60 ml), 0.3 M (80ml) (product fractions) and 0.6 M (80 ml) (fraction of free carboxylicacid form of Alexa fluor 488) TEAB. 0.1 M eluent was discarded. 0.3 Meluent was collected and evaporated under reduced pressure. The residuewas co-evaporated with water (2×10 ml) and then further purified bysemi-preparative reverse phase HPLC [HPLC gradient: A, 100% 0.1 M TEAB;B 100% MeCN; 0-2 min, 2% B (flow 2-5 ml/min); 2-20 min, 2-20% B (flow 5ml/min); 20-22 min, 20-95% B (flow 5 ml/min); 22-25 min, 95% B (flow 5ml/min); 25-27 min, 95-2% B (flow 5 ml/min); 27-30 min, 2% B (flow 5-2ml/min)]. The first isomer product with retention time of 19.11 min wascollected and evaporated under reduced pressure and the residue wasco-evaporated with water (2×5 ml) to give the title compound as triethylammonium salt (5.22 mmol, quantification at λ_(max(493)) in 0.1 M TEABbuffer, 16.8%). This isomer was used to forward synthesis. The secondisomer product with retention time 19.73 min was kept aside. ¹HNMR ofproduct with Rt 19.11 min in D₂O indicated approximately twotriethylammonium count ions. ¹H NMR (400 MHz, D₂O), δ 1.11 (t, J=7.3 Hz,18H, CH₃, triethylammonium count ion), 2.37 (t, J=6.4 Hz, 2H, CH₂), 3.03(q, J=7.3 Hz, 12H, CH₂, triethylammonium count ion), 3.30-3.70 (m, 50H),6.80 (d, J=9.3 Hz, 2H, Ar—H), 7.08 (d, J=9.3 Hz, 2H, Ar—H), 7.53 (s, 1H,Ar—H) and 7.80-7.95 (m, 2H, Ar—H). LC-MS (electrospray negative): 565.85[(M/2e)−1].

Alexa488-PEG₁₂-LN₃ linker carboxylic acid (2)

Alexa488-PEG₁₂ carboxylic acid (1) (5 μmolo) was co-evaporated with dryDMF (5 ml) and the residue was then dissolved in dry DMF (1.5 ml). Asolution of TSTU (10 μmol, 100 μl, concentration: 30 mg TSTU in 1 ml dryDMF) in dry DMF was added. The reaction was stirred at room temperaturefor 10 minutes. LN₃ linker((2-{2-[3-(2-amino-ethylcarbamoyl)-phenoxy]-1-azido-ethoxy}-ethoxy)-aceticacid) (15 μmol, 5.51 mg) was added followed by DIPEA (50 μmol, 8.7 μl).The reaction was stirred at room temperature overnight. The reaction wasthen diluted with 0.1 M TEAB buffer (10 ml) and then loaded onto aDEAE-A25 Sephadex column (1×10 cm). The column was eluted with 0.1 MTEAB (60 ml, this fraction was discarded) and 0.35 M TEAB (80 ml). Theproduct-containing fraction (0.35 M eluent) was evaporated under reducedpressure. The residue was co-evaporated with water (2×10 ml). Theresidue was further purified by semi-preparative reverse phase HPLC[HPLC gradient: A, 100% 0.1 M TEAB; B 100% MeCN; 0-2 min, 5% B (flow 2-5ml/min); 2-15 min, 5-22% B (flow 5 ml/min); 15-21 min, 22-45% B (flow 5ml/min); 21-22 min, 45-95% B (flow 5 ml/min); 22-25 min, 95% B (flow 5ml/min); 25-27 min, 95-5% B (flow 5 ml/min); 27-30 min, 5% B (flow 5-2ml/min)]. The product with retention time of 19.26 min was collected andevaporated under reduced pressure and the residue was co-evaporated withwater (2×5 ml) to give the title compound (2) as triethyl ammonium salt(2.29 μmol, quantification at μ_(max(494)) in 0.1 M TEAB buffer, 45.8%).¹HNMR of product with Rt 19.26 min in D₂O indicated approximatelyaverage 5.6 triethylammonium count ions. ¹H NMR (400 MHz, D₂O), δ 1.00(t, J=7.3 Hz, 51H, CH₃, triethylammonium count ion), 2.35 (t, J=5.6 Hz,2H, CH₂), 2.75 (q, J=7.3 Hz, 34H, CH₂, triethylammonium count ion),3.22-3.62 (m, 56H), 3.70-3.78 (m, 1H), 3.79 (s, 2H, OCH₂CO₂H), 3.87-3.95(m, 1H), 4.02-4.12 (m, 2H, ArOCH₂), 4.90-4.98 (m, 1H, CHN₃), 6.78 (d,J=9.3 Hz, 2H, Ar—H), 6.98-7.08 (m, 3H, Ar—H), 7.14-7.22 (m, 2H, Ar—H),7.28 (t, J=7.9 Hz, 1H, Ar—H), 7.51 (s, 1H, Ar—H), 7.86 (d, J=8.0 Hz, 1H,Ar—H) and 7.91 (d, J=8.0 Hz, 1H, Ar—H). LC-MS (electrospray negative):740.35 [(M/2e)−1], 493.50 [(M/3e)−1] as mono-potassium adduct salt.

7-[3-(-Alexa488-PEG₁₂-LN₃-linkeracetylamino)-prop-1-ynyl]-3′-azidomethyl-dGTP(3)

Alexa488-PEG₁₂-LN₃ linker carboxylic acid (2) (2 μmol) was co-evaporatedunder reduced pressure with anhydrous DMF (2 ml) and the re-dissolved inanhydrous DMF (0.8 ml). A solution of TSTU (6 μmol, 100 μl,concentration: 18 mg TSTU in 1 ml dry DMF) in dry DMF was added. Thereaction was stirred at room temperature for 10 minutes.[7-(3-amino-prop-1-ynyl)]-3′-azidomethyl-dGTP (6 μmol, prepared byevaporating an aqueous solution of[7-(3-amino-prop-1-ynyl)]-3′-azidomethyl-dGTP in 0.1 M TEAB buffer (1.82ml) and with tri-n-butyl amine (14.3 μl, 60 μmol) in DMF (200 μl)) in0.1 M TEAB buffer (0.2 ml) was then added. The reaction was stirred atroom temperature for 4 hrs, and then diluted with chilled 0.1 M TEAB (4ml). The whole reaction mixture was then loaded onto a DEAE-A25 Sephadexcolumn (1×10 cm). The column was then eluted with 0.1 M (60 ml), 0.3 M(60 ml) and 0.7 M (80 ml) TEAB buffer. The 0.7 M eluent was collectedand evaporated under reduced pressure and the residue was co-evaporatedwith water (2×5 ml). The residue was dissolved in 0.1 M TEAB (5 ml),then further purified by semi-preparative reverse phase HPLC [HPLCgradient: A, 100% 0.1 M TEAB; B 100% MeCN; 0-2 min, 5% B (flow 2-5ml/min); 2-15 min, 5-22% B (flow 5 ml/min); 15-21 min, 22-25% B (flow 5ml/min); 21-22 min, 25-95% B (flow 5 ml/min); 22-25 min, 95% B (flow 5ml/min); 25-27 min, 95-5% B (flow 5 ml/min); 27-30 min, 5% B (flow 5-2ml/min)]. The product with retention time of 18.49 min was collected andevaporated under reduced pressure to give the title compound (3) astriethyl ammonium salt (1.27 μmol, quantification at λ_(max(494)) in 0.1M TEAB buffer, 63.5%). ¹HNMR of product with Rt 18.49 min in D₂Oindicated approximately average 71 triethylammonium count ions. ¹H NMR(400 MHz, D₂O), δ 1.09 (t, J=7.3 Hz, 639H, CH₃, triethylammonium countion), 2.20-2.31 (m, 1H, H_(a)-2′), 2.32 (t, J=5.9 Hz, 2H, CH₂),2.36-2.52 (m, 1H, H_(b)-2′), 3.01 (q, J=7.3 Hz, 426H, CH₂,triethylammonium count ion), 3.23-3.59 (m, 54H), 3.61-3.82 (m, 3H),3.87-4.12 (m, 9H), 4.13-4.18 (m, 1H, H-4′), 4.44-4.50 (m, 1H, H-3′),4.74-4.80 (m, 2H, OCH₂N₃), 4.89-4.97 (m, 1H, CHN3), 5.96-6.08 (m, 1H,H-1′), 6.73-6.81 (m, 3H), 6.95 (s, 1H), 7.00-7.15 (m, 5H), 7.49 (s, 1H,Ar—H), and 7.80-7.92 (m, 2H, Ar—H). ³¹P NMR (D₂O), δ −20.94 (m, ^(β)P),−10.08 (d, J=18.6 Hz, ^(β)P) and −5.00 (d, J=21.1 Hz, ^(γ)P). LC-MS(electrospray negative): 1038.2 [(M/2e)−1].

Example 2 Preparation of7-[3-(-Alexa488-PEG₂₄-LN₃-linkeracetylamino)-prop-1-ynyl]-3′-azidomethyl-dGTP(6)

Alexa488-PEG₂₄ carboxylic acid (4)

Alexa fluor 488 carboxylic acid succinimidyl ester (mixed isomers, 10mg, 15.5 μmoles) was dissolved in dry DMF (2 ml). Amino dPEG₂₄ t-butylester (41.5 mg, 34.5 μmoles) and DIPEA (69.6 μl, 400 μmoles) were added.The reaction was then stirred at RT for 3 hrs. All the solvents wereevaporated under reduced pressure and the residue was then dissolved ina mixture solvent of TFA (2 ml) and DCM (8 ml). The reaction was stirredat room temperature. After 30 minutes, all the solvents were evaporatedunder reduce pressure. The residue was then diluted with chilled TEABbuffer (0.1 M, 100 ml). The solution was then loaded onto a column ofDEAE-A25 Sephadex (2×15 cm). The column was eluted with 0.1 M (50 ml)and 0.3 M (50 ml) TEAB buffer. 0.1 M eluent was discarded. 0.3 M eluentwas collected and evaporated under reduced pressure. The residue wasco-evaporated with water (2×10 ml) and then further purified bysemi-preparative reverse phase HPLC [HPLC gradient: A, 100% 0.1 M TEAB;B 100% MeCN; 0-2 min, 5% B (flow 2-5 ml/min); 2-20 min, 5-25% B (flow 5ml/min); 20-22 min, 25-95% B (flow 5 ml/min); 22-25 min, 95% B (flow 5ml/min); 25-27 min, 95-5% B (flow 5 ml/min); 27-30 min, 5% B (flow 5-2ml/min)]. The first isomer product with retention time of 20.25 min wascollected and evaporated under reduced pressure and the residue wasco-evaporated with water (2×5 ml) to give the title compound as triethylammonium salt (5.5 μmol, quantification at λ_(max(494)) in 0.1 M TEABbuffer, 35.4%). This isomer was used to forward synthesis. The secondisomer product with retention time 20.62 min was kept aside. ¹HNMR ofproduct with Rt 20.25 min in D₂O indicated approximately 1.5triethylammonium count ions. ¹H NMR (400 MHz, D₂O), δ 1.12 (t, J=7.3 Hz,13.5H, CH₃, triethylammonium count ion), 2.46 (t, J=6.2 Hz, 2H, CH₂),3.04 (q, J=7.3 Hz, 9H, CH₂, triethylammonium count ion), 3.34-3.66 (m,98H), 6.81 (d, J=9.3 Hz, 2H, Ar—H), 7.08 (d, J=9.3 Hz, 2H, Ar—H), 7.55(s, 1H, Ar—H) and 7.93 (s, 2H, Ar—H). LC-MS (electrospray negative):829.75 [(M/2e)−1], 553.25 [(M/3e)−1].

Alexa488-PEG₂₄-LN₃ linker carboxylic acid (5)

Alexa488-PEG₂₄ carboxylic acid (4) (0.6 μmol) was co-evaporated with dryDMF (2 ml) and the residue was then dissolved in dry DMF (1 ml). Asolution of TSTU (2.4 μmol, 100 μl, concentration: 7.23 mg TSTU in 1 mldry DMF) in dry DMF was added. The reaction was stirred at roomtemperature for 10 minutes. LN₃ linker((2-{2-[3-(2-amino-ethylcarbamoyl)-phenoxy]-1-azido-ethoxy}-ethoxy)-aceticacid) (6 μmol, 2.2 mg) was added followed by DIPEA (30 μmol, 5.2 μl).The reaction was stirred at room temperature. After overnight (18 hrs),the reaction was diluted with chilled 0.1 M TEAB buffer (10 ml) and thenloaded onto a DEAE-A25 Sephadex column (1×10 cm). The column was elutedwith 0.1 M TEAB (30 ml, this fraction was discarded) and 0.30 M TEAB (50ml). The product-containing fraction (0.30 M eluent) was evaporatedunder reduced pressure. The residue was co-evaporated with water (2×5ml). The residue was further purified by semi-preparative reverse phaseHPLC [HPLC gradient: A, 100% 0.1 M TEAB; B 100% MeCN; 0-2 min, 5% B(flow 2-5 ml/min); 2-15 min, 5-22% B (flow 5 ml/min); 15-21 min, 22-50%B (flow 5 ml/min); 21-22 min, 50-95% B (flow 5 ml/min); 22-25 min, 95% B(flow 5 ml/min); 25-27 min, 95-5% B (flow 5 ml/min); 27-30 min, 5% B(flow 5-2 ml/min)]. The product with retention time of 19.46 min wascollected and evaporated under reduced pressure and the residue wasco-evaporated with water (2×2 ml) to give the title compound (5) astriethyl ammonium salt (0.224 μmol, quantification at λ_(max(494)) in0.1 M TEAB buffer, 37.3%). ¹HNMR of product with Rt 19.46 min in D₂Oindicated approximately average one triethylammonium count ions. ¹H NMR(400 MHz, D₂O), δ 1.11 (t, J=7.3 Hz, 9H, CH₃, triethylammonium countion), 2.35 (t, J=5.9 Hz, 2H, CH₂), (q, J=7.3 Hz, 6H, CH₂,triethylammonium count ion), 3.20-3.70 (m, 104H), 3.65-3.78 (m, 1H),3.80 (s, 2H, OCH₂CO₂H), 3.85-4.05 (m, 1H), 4.13 (d, J=4.2 Hz, 2H,ArOCH₂), 4.96 (t, J=4.3 Hz, 1H, CHN₃), 6.80 (d, J=9.4 Hz, 2H, Ar—H),7.06 (s, 1H, Ar—H), 7.08 (d, J=9.4 Hz, 2H, Ar—H), 7.15-7.25 (m, 2H,Ar—H), 7.32 (t, J=7.8 Hz, 1H, Ar—H), (s, 1H, Ar—H), 7.87 (d, J=8.2 Hz,1H, Ar—H) and 7.91 (d, J=8.2 Hz, 1H, Ar—H). LC-MS (electrospraynegative): 669.30 [(M/3e)−1], 501.90 [(M/4e)−1].

7-[3-(-Alexa488-PEG₂₄-LN₃-linkeracetylamino)-prop-1-ynyl]-3′-azidomethyl-dGTP(6)

Alexa488-PEG₂₄-LN₃ linker carboxylic acid (5) (0.5 μmol) wasco-evaporated under reduced pressure with anhydrous DMF (1 ml) and thenre-dissolved in anhydrous DMF (0.5 ml). A solution of TSTU (2 μmol, 50μl, concentration: 12 mg TSTU in 1 ml dry DMF) in dry DMF was added. Thereaction was stirred at room temperature for 10 minutes.[7-(3-amino-prop-1-ynyl)]-3′-azidomethyl-dGTP (2.5 μmol) in 0.1 M TEABbuffer (0.2 ml) was then added. The reaction was stirred at roomtemperature for 3 hrs and stored in fridge overnight (18 hrs). It wasthen diluted with chilled 0.1 M TEAB (10 ml). The whole reaction mixturewas then loaded onto a DEAE-A25 Sephadex column (1×10 cm). The columnwas then eluted with 0.1 M (30 ml), 0.3 M (30 ml) and 0.6 M (50 ml) TEABbuffer. The 0.6 M eluent was collected and evaporated under reducedpressure and the residue was co-evaporated with water (10 ml). Theresidue was dissolved in 0.1 M TEAB (5 ml), then further purified bysemi-preparative reverse phase HPLC [HPLC gradient: A, 100% 0.1 M TEAB;B 100% MeCN; 0-2 min, 5% B (flow 2-5 ml/min); 2-15 min, 5-25% B (flow 5ml/min); 15-21 min, 25-30% B (flow 5 ml/min); 21-22 min, 30-95% B (flow5 ml/min); 22-25 min, 95% B (flow 5 ml/min); 25-27 min, 95-5% B (flow 5ml/min); 27-30 min, 5% B (flow 5-2 ml/min)]. The product with retentiontime of 19.57 min was collected and evaporated under reduced pressure togive the title compound (6) as triethyl ammonium salt (0.231 μmol,quantification at λ_(max(494)) in 0.1 M TEAB buffer, 46.2%). ¹HNMR ofproduct with Rt 19.57 min in D₂O indicated approximately average onetriethylammonium count ions. ¹H NMR (400 MHz, D₂O), δ 1.11 (t, J=7.3 Hz,9H, CH₃, triethylammonium count ion), 2.20-2.24 (m, 1H, H_(a)-2), 2.35(t, J=5.8 Hz, 2H, CH₂), 2.38-2.48 (m, 1H, H_(b)-2′), 3.03 (q, J=7.3 Hz,6H, CH₂, triethylammonium count ion), 3.40-3.60 (m, 102H), 3.65-3.85 (m,3H), 3.87-4.11 (m, 9H), 4.12-4.20 (m, 1H, H-4′), 4.49-4.51 (m, 1H,H-3′), 4.77-4.82 (m, 2H, OCH₂N₃), 4.85-5.00 (m, 1H, CHN₃), 6.00-6.20 (m,1H, H-1′), 6.73-6.85 (m, 3H), 6.96 (s, 1H), 7.05-7.18 (m, 5H), 7.52 (d,J=1.4 Hz 1H, Ar—H), 7.80 (d, J=8.1 Hz, 1H, Ar—H) and 7.90 (dd, J=1.5 and8.1 Hz, 1H, Ar—H). LC-MS (electrospray negative): 1302.9 [(M/2e)−1].

Comparative Example Synthesis of 7-[3-(-Alexa488-LN₃-linkeracetylamino)-prop-1-ynyl]-3′-azidomethyl-dGTP (8)

Alexa488-LN₃ linker carboxylic acid (7)

Alexa flour 488 6-carboxylic acid (9 μmol) was stirred withN,N′-di-succinimidyl carbonate (19.8 μmol, 5.07 mg), DMAP (19.8 μmol,2.42 mg) and DIPEA (30 μmol, 5.23 μl) in dry DMF (1 ml). After 15minutes at room temperature, LN₃ linker((2-{2-[3-(2-amino-ethylcarbamoyl)-phenoxy]-1-azido-ethoxy}-ethoxy)-aceticacid) (30 μmol, 11.0 mg) was added followed by DIPEA (60 μmol, 10.45μl). The reaction was stirred at room temperature overnight (18 hrs).The reaction was then diluted with chilled water (15 ml) and then loadedonto a DEAE-A25 Sephadex column (1×10 cm). The column was eluted with0.1 M TEAB (50 ml, this fraction was discarded) and 1.0 M TEAB (50 ml).The product-containing fraction (1.0 M eluent) was evaporated underreduced pressure. The residue was co-evaporated with water (2×10 ml).The residue was further purified by preparative HPLC [HPLC gradient: A,100% 0.1 M TEAB; B 100% MeCN; 0-2 min, 5% B (flow 2-10 ml/min); 2-19min, 5-25% B (flow 10 ml/min); 19-21 min, 25-95% B (flow 10 ml/min);21-24 min, 95% B (flow 10 ml/min); 24-26 min, 95-5% B (flow 10 ml/min);26-30 min, 5% B (flow 10-2 ml/min)]. The product with retention time of20.06 min was collected and evaporated under reduced pressure and theresidue was co-evaporated with water (2×5 ml) to give the title compound(7) as triethyl ammonium salt (3.69 μmol, quantification at λ_(max(495))in 0.1 M TEAB buffer, 51%; also recovered 1.76 μmole Alexa flour 4886-carboxylic acid). ¹HNMR of product in D₂O indicated approximatelyaverage 3.7 triethylammonium count ions. ¹H NMR (400 MHz, D₂O), δ 1.08(t, J=7.3 Hz, 33H, CH₃, triethylammonium count ion), 2.94 (q, J=7.3 Hz,22H, CH₂, triethylammonium count ion), 3.45-3.65 (m, 6H), 3.68-3.78 (m,1H), 3.79 (s, 2H, OCH₂CO₂H), 3.87-3.93 (m, 2H), 3.95-4.05 (m, 1H), 4.84(t, J=4.0 Hz, 1H, CHN₃), 6.69 (d, J=9.3 Hz, 1H, Ar—H), 6.72 (d, J=9.3Hz, 1H, Ar—H), 6.84 (d, J=9.3 Hz, 1H, Ar—H), 6.85-6.94 (m, 2H, Ar—H),6.95-7.04 (m, 2H, Ar—H), 7.07 (t, J=7.9 Hz, 1H, Ar—H), 7.13 (s, 1H,Ar—H), 7.81 (d, J=8.1 Hz, 1H, Ar—H) and 7.84 (d, J=8.1 Hz, 1H, Ar—H).LC-MS (electrospray negative): 882.80 [M-1].

7-[3-(-Alexa488-LN₃-linkeracetylamino)-prop-1-ynyl]-3′-azidomethyl-dGTP(8)

Alexa flour 488 LN₃ linker carboxylic acid (7) (1.65 μmol) was dissolvedin dry DMF (0.5 ml). N,N′-di-succinimidyl carbonate (5.4 μmol, 1.38 mg)and DMAP (3.6 μmol, 0.44 mg) were added. After 15 minutes at roomtemperature, all the above reaction mixture was added to[7-(3-amino-prop-1-ynyl)]-3′-azidomethyl-dGTP (5.8 μmol, prepared byevaporating an aqueous solution of[7-(3-amino-prop-1-ynyl)]-3′-azidomethyl-dGTP in 0.1 M TEAB buffer (1.45ml) and with tri-n-butyl amine (144 μl)). The reaction was stirred atroom temperature 3 hrs. The reaction was then diluted with chilled 0.1 MTEAB (10 ml) and then loaded onto a DEAE-A25 Sephadex column (1×8 cm).The column was eluted with 0.1 M (50 ml, this fraction was discarded)and 2.0 M TEAB (50 ml). The product-containing fraction (2.0 M eluent)was evaporated under reduced pressure. The residue was co-evaporatedwith water (2×5 ml). The residue was further purified bysemi-preparative reverse phase HPLC [HPLC gradient: A, 100% 0.1 M TEAB;B 100% MeCN; 0-2 min, 5% B (flow 2-5 ml/min); 2-14 min, 5-20% B (flow 5ml/min); 14-20 min, 20-23% B (flow 5 ml/min); 20-22 min, 23-95% B (flow5 ml/min); 22-25 min, 95% B (flow 5 ml/min); 25-26 min, 95-5% B (flow 5ml/min); 26-30 min, 5% B (flow 5-2 ml/min)]. The product with retentiontime of 16.63 min was collected and evaporated under reduced pressureand the residue was co-evaporated with water (5 ml) to give the titlecompound (8) as triethyl ammonium salt (0.257 μmol, quantification atλ_(max(495)) in 0.1 M TEAB buffer, 15.6%). ¹H NMR (400 MHz, D₂O),2.14-2.30 (m, 1H, H_(a)-2′), 2.38-2.52 (m, 1H, H_(b)-2′), 3.49-4.02 (m,16H), 4.15-4.25 (m, 1H), 4.45-4.55 (m, 1H), 4.80 (d, J=6.9 Hz, 1H,Ar—OCH_(a)H_(b)), 4.84 (d, J=6.9 Hz, 1H, Ar—OCH_(a)H_(b)), 4.87-4.92 (m,1H, CHN₃), 5.86-5.95 (m, 1H), 6.54 (t, J=9.1 Hz, 1H, Ar—H), 6.62-6.67(m, 1H, Ar—H), 6.70 (d, J=8.0 Hz, 1H, Ar—H), 6.78-6.83 (m, 2H, Ar—H),6.87-7.04 (m, 4H, Ar—H), 7.39 (s, 1H, Ar—H) and 7.81-7.90 (m, 2H, Ar—H).MS (electrospray negative): 757.40 [(M/2e)−1], 505.00 [(M/3e)−1] (asmono potassium adduct salt).

Example 3 Preparation of Atto532-Peg12-LN3-dGTP

Step 1

Synthesis of Atto532-Peg12

Atto532NHS ester (20 mg, 26.9 μmol) (Atto-tec AD532-3) was dissolved inDMF (1.5 ml). A solution of H₂N-PEG12-COOH (49.8 mg, 80.7 μmol) in 0.1 MTEAB (0.5 ml) was added to the reaction. The reaction was monitored byTLC (eluting system ACN: H₂O 4:1) and reached completion in 90 min. Itwas quenched with 2 ml of 0.1 M TEAB and concentrated to dryness. Thecrude was purified by doing a Sephadex column (1×10 cm). We eluted threefractions, first with 40 ml of 0.1 M TEAB, second with 100 ml of 0.3 MTEAB and finally with 100 ml of 0.5 M TEAB. The product of the reactionwas contained in fraction 2. This was submitted to HPLC purification(5-50 method in 20 min in the semiprep Zorbax column), retention time13.7 min. The product was obtained in 64% yield.

MS (es-, m/z): 1243, 622

1H NMR (400 MHz; D₂O) 7.65-7.56 (2H, m, CHar, CHar), 7.52-7.45 (1H, m,CHar), 7.40-7.36 (1H, m, CHar), 7.23-7.18 (2H, m which includes doublet,J 9.6, CHar, CHar), 6.92 (1H, d, J 9.6 CHar), 6.91 (1H, d, J 9.6, CHar),3.58 (2H, t, J 6.8, CH₂), 3.55-3.45 [44H, m, 11x(O—CH₂)+11x(CH₂—O)],3.40 (2H, t, J 5.6, CH₂), 3.33 (4H, q, J 7.2, 2xCH₂), 3.19 (1H, t, J5.6, CH), 3.14 (1H, t, J 5.6, CH), 3.09 (1H, br.t, CH), 2.78 (3H, s,CH₃), 2.31 (2H, t, J 6.8, CH₂), 1.60-1.52 (2H, m, CH₂), 1.32-1.24 (2H,m, CH₂), 1.17 (6H, t, J 7.2, 2xCH₃).

Step 2

Preparation of Atto532-PEG12-LN3

Atto532PEG (21.6 mg, 17.4 μmol) was dissolved in DMF (1.8 ml). Asolution of TSTU (7.8 mg, 26.1 μmol) in DMF was added to the reaction.Since not much progress was observed after 30 min by TLC (eluting systemACN: H₂O 4:1), DIPEA (15 μl, 87 μmol) was added. The activation wascompleted in 30 min and LN3 (15.9 mg, 43.5 μmol) dissolved in DMF wasadded. The reaction was left stirring for 16 h, after which it wasquenched with 10 ml of 0.1 M TEAB and vacuumed off. The reaction crudewas purified by HPLC (5-50 method in 20 min in the semiprep Zorbaxcolumn), retention time 14.9 min. The product was obtained in 66% yield.

MS (es-, m/z): 796

1H NMR (400 MHz; D₂O) 7.66-7.56 (2H, m, CHar, CHar), 7.54-7.45 (1H, m,CHar), 7.38-7.34 (1H, m, CHar), 7.28 (1H, q, J 8.0, CHar), 7.23-7.15(4H, m, CHar), 7.08-7.01 (1H, br.d, J 8.0, CHar), 6.90 (1H, d, J 5.2,CHar), 6.88 (1H, d, J 5.2, CHar), 4.94 (1H, t, J 4.4, CHN₃), 4.12 (2H,br.d, J 4.0, CH₂), 4.00-3.86 (2H, double m, CHH), 3.81 (2H, s, O—CH₂),3.81-3.72 (1H, m, CHH), 3.63-3.55 (5H, m, 2xCH₂+CH), 3.54-3.26 [53H,triple m, 4xCH₂+CH+11x(O—CH₂)+11x(CH₂—O)], (2H, m, CH₂), 2.76 (3H, s,CH₃), 2.35 (2H, t, J 5.6, CH₂), 1.96-1.90 (1H, m, CH), 1.60-1.49 (1H, m,CH), (1H, m, CH₂), 1.17 (6H, t, J 7.2, 2xCH₃).Step 3Synthesis of G-Atto532-PEG12-LN3

Atto532PEGLN3 (18 mg, 11.3 μmol) was dissolved in DMF (3 ml). A solutionof TSTU (5.1 mg, 17 μmol) in DMF (200 μl) was added. The progress of thereaction was monitored by TLC (eluting system ACN: H2O 4:1). Noactivation is observed after 30 min, so DIPEA (10 μl) was added. After30 min, the TLC shows that the activation was completed. PPPG (34 μmol,2.25 mM) was co-evaporated with tributylamine (81 μl) and redissolved in0.1 M TEAB (0.5 ml). After 30 min, TLC showed that the reaction had goneto completion (eluting system ACN: H2O 4:1). The reaction was quenchedwith 10 ml of 0.1 M TEAB at 0° C. and vacuumed off. The reaction crudewas purified by HPLC (5-50 method in 20 min in the semiprep Zorbaxcolumn), retention time 14.8 min. The product was obtained in 57% yield.

MS (es-, m/z): 1095, 729, 546

1H NMR (400 MHz; D₂O) 7.64-7.60 (2H, m, CHar), 7.51-7.44 (1H, m, CHar),7.35-7.31 (1H, m, CHar), 7.16-7.12 (4H, m, CHar), 7.09 (1H, s, CHbase),6.99-6.96 (1H, br.s, CHar), 6.88 (1H, d, J 4.0, CHar), 6.86 (1H, d, J4.0, CHar), 6.83-6.77 (1H, m, CHar), 6.06-5.96 (1H, m, H-1′), 4.96 (1H,br.s, CHHN₃), 4.82 (1H, br.s, CHHN₃), 4.54-4.46 (1H, m, H-3′), 4.20-4.14(1H, m, H-4′), 4.12-3.89 (8H, double m, 3xCH₂+2H-5′), 3.86-3.60 (4H, m,CH₂+CH₂—N), 3.56 (2H, t, J 6.0, CH₂), 3.54-3.28 [56H, set m,11x(O—CH₂)+11x(CH₂—O)+6CH₂], 3.18 (2H, t, J 5.6, CH₂), 2.74 (3H, s,CH₃), 2.46-2.23 (4H, m+t+m, J 6.0, 2H-2′+CH₂), 1.61-1.34 (3H, m,CH₂+CH), 1.29-1.14 (8H, m+t, J 7.2, CH₂+2xCH₃).

Example 4 Demonstration of Reduced Quenching of Fluorophores in ModifiedNucleotides of the Invention

The modified nucleotides of Examples 1 and 2 ((3) and (6)) and thecompound of the Comparative Example (8) described above were eachincorporated into a polynucleotide by phosphodiester linkage of themodified nucleotide to the 3′ end of a DNA strand, the precise sequenceof which is not of relevance. The fluorescent intensity of the Alexa 488dye in the modified nucleotides was then measured, both before and aftertreatment with Tris-(2-carboxyethyl) phosphine (TCEP). FIG. 1 shows theintensity measured of the Alexa 488 dye, for all three modifiednucleotides, both before and after cleavage of the linkers with TCEP.The modified nucleotide of the comparative example (i.e. G-N-3-A488 withno PEG in the linker) clearly shows the highest level of quenching (i.e.lowest fluorescence intensity) before TCEP cleavage. However, thesimilarity in fluorescence intensity measured after cleavage of allthree linkers is striking. Since the point of cleavage in the chainleaves the PEG moieties still attached to the Alexa 488 fluorophore,this experiment demonstrates that because the “free” fluorophore (i.e.without the guanine base) is not quenched in solution, the enhancedsignal in the fully functionalised nucleotides (ff's) of the inventionis not simply an artefact of the PEG moiety being attached to thefluorophore. The FIGURE also illustrates that compound (6) demonstratesa greater reduction in quenching (i.e. higher fluorescence intensitybefore TCEP treatment) over not only the modified nucleotides of thecomparative example, but also over compound (3).

All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. While thisinvention has been particularly shown and described with references topreferred embodiments, it will be understood by those skilled in the artthat various changes in form and details may be made without departingfrom the scope of the invention encompassed by the claims.

1. A modified nucleotide consisting of a guanine base or a7-deazaguanine base attached to a fluorophore through a linking group,one or more phosphate groups, and a ribose or 2′-deoxyribose having ablocking group attached to the 3′ oxygen atom thereof, wherein saidlinking group comprises a spacer group of formula —((CH₂)₂O)_(n)—wherein n is an integer between 10 and 30, and wherein the presence ofthe linking group reduces fluorescent signal quenching of thefluorophore by the guanine base or 7-deazaguanine base.
 2. The modifiednucleotide of claim 1 wherein n is between 10 and
 25. 3. The modifiednucleotide of claim 1 wherein said linking group is cleavable.
 4. Themodified nucleotide of claim 3 wherein said linking group and saidblocking group are cleavable.
 5. The modified nucleotide of claim 1which is a deoxyribonucleotide.
 6. A method of detecting a nucleotideincorporated into a polynucleotide which comprises: (a) incorporating atleast one modified nucleotide as defined in claim 1 into apolynucleotide, wherein said incorporating refers to joining themodified nucleotide to the free 3′ hydroxyl group of a nucleic acidstrand via formation of a phosphodiester linkage and (b) detecting thenucleotide(s) incorporated into the polynucleotide by detecting thefluorescent signal from said incorporated nucleotide(s).
 7. A methodaccording to claim 6 wherein step (a) comprises incorporating at leastone modified nucleotide into a polynucleotide using a polymerase enzyme.8. A method of sequencing a template nucleic acid molecule comprising:incorporating one or more modified nucleotides of claim 1 into a strandof nucleic acid complementary to the template nucleic acid anddetermining the identity of the base present in one or more incorporatednucleotide(s) in order to determine the sequence of the template nucleicacid molecule; wherein the identity of the base present in saidnucleotide(s) is determined by detecting a fluorescent signal producedby said nucleotide(s).
 9. A method according to claim 8 wherein theidentity of the base present in said nucleotide(s) is determined aftereach nucleotide incorporation step.
 10. A modified nucleotide consistingof a guanine base or a 7-deazaguanine base attached to a fluorophorethrough a linking group, one or more phosphate groups, and a ribose,2′-deoxyribose or 2′, 3′ dideoxyribose, wherein said linking groupcomprises a spacer group of formula —((CH₂)₂O)_(n)— wherein n is aninteger between 10 and 30, and wherein the presence of the linking groupreduces fluorescent signal quenching of the fluorophore by the guaninebase or 7-deazaguanine base.
 11. A method of detecting a nucleotideincorporated into a polynucleotide which comprises: (a) incorporating atleast one modified nucleotide as defined in claim 10 into apolynucleotide, wherein said incorporating refers to joining themodified nucleotide to the free 3′ hydroxyl group of a nucleic acidstrand via formation of a phosphodiester linkage and (b) detecting thenucleotide(s) incorporated into the polynucleotide by detecting thefluorescent signal from said incorporated nucleotide(s).
 12. The methodaccording to claim 11 wherein step (a) comprises incorporating at leastone modified nucleotide into a polynucleotide using a polymerase enzyme.13. The method according to claim 11 wherein n is between 10 and
 25. 14.The method according to claim 11 wherein said linking group iscleavable.